The present invention relates to isolated and purified nucleic acids and encoded proteins from the genera Renilla, Gaussia, Philocarpus and Pleuromamma. More particularly, nucleic acids encoding luciferase and fluorescent proteins from species of these genera are provided.
Luminescence is a phenomenon in which energy is specifically channeled to a molecule to produce an excited state. Return to a lower energy state is accompanied by release of a photon (hxcex3). Luminescence includes fluorescence, phosphorescence, chemiluminescence and bioluminescence. Bioluminescence is the process by which living organisms emit light that is visible to other organisms. Luminescence may be represented as follows:
A+Bxe2x86x92X*+Y
X*xe2x86x92X+hv,
where X* is an electronically excited molecule and hxcex3 represents lightemission upon return of X* to a lower energy state. Where the luminescence is bioluminescence, creation of the excited state derives from an enzyme catalyzed reaction. The color of the emitted light in a bioluminescent (or chemiluminescent or other luminescent) reaction is characteristic of the excited molecule, and is independent from its source of excitation and temperature.
An essential condition for bioluminescence is the use of molecular oxygen, either bound or free in the presence of a luciferase. Luciferases, are oxygenases, that act on a substrate, luciferin, in the presence of molecular oxygen and transform the substrate to an excited state. Upon return to a lower energy level, energy is released in the form of light [for reviews see, e.g., McElroy et al. (1 966) in Molecular Architecture in Cell Physiology, Hayashi et al., eds., Prentice-Hall, Inc., Englewood Cliffs, N.J., pp. 63-80; Ward et al., Chapter 7 in Chemi-and Bioluminescence, Burr, ed., Marcel Dekker, Inc. NY, pp.321-358; Hastings, J. W. in (1995) Cell Physiology:Source Book, N. Sperelakis (ed.), Academic Press, pp 665-681; Luminescence, Narcosis and Life in the Deep Sea, Johnson, Vantage Press, NY, see, esp. pp. 50-56].
Though rare overall, bioluminescence is more common in marine organisms than in terrestrial organisms. Bioluminescence has developed from as many as thirty evolutionarily distinct origins and, thus, is manifested in a variety of ways so that the biochemical and physiological mechanisms responsible for bioluminescence in different organisms are distinct. Bioluminescent species span many genera and include microscopic organisms, such as bacteria [primarily marine bacteria including Vibrio species], fungi, algae and dinoflagellates, to marine organisms, including arthropods, mollusks, echinoderms, and chordates, and terrestrial organism including annelid worms and insects.
Assays Employing Bioluminescence
During the past twenty years, high-sensitivity biochemical assays used in research and in medicine have increasingly employed luminescence and fluorescence rather than radioisotopes. This change has been driven partly by the increasing expense of radioisotope disposal and partly by the need to find more rapid and convenient assay methods. More recently, the need to perform biochemical assays in situ in living cells and whole animals has driven researchers toward protein-based luminescence and fluorescence. The uses of firefly luciferase for ATP assays, aequorin and obelin as calcium reporters, Vargula luciferase as a neurophysiological indicator, and the Aequorea green fluorescent protein as a protein tracer and pH indicator show the potential of bioluminescence-based methods in research laboratories.
Bioluminescence is also beginning to directly impact medicine and biotechnology; for example, Aequorea GFP is employed to mark cells in murine model systems and as a reporter in high throughput drug screening. Renilla luciferase is under development for use in diagnostic platforms.
Bioluminescence Generating Systems
Bioluminescence, as well as other types of chemiluminescence, is used for quantitative determinations of specific substances in biology and medicine. For example, luciferase genes have been cloned and exploited as reporter genes in numerous assays, for many purposes. Since the different luciferase systems have different specific requirements, they may be used to detect and quantify a variety of substances. The majority of commercial bioluminescence applications are based on firefly [Photinus pyralis] luciferase. One of the first and still widely used assays involves the use of firefly luciferase to detect the presence of ATP. It is also used to detect and quantify other substrates or co-factors in the reaction. Any reaction that produces or utilizes NAD(H), NADP(H) or long chain aldehyde, either directly or indirectly, can be coupled to the light-emitting reaction of bacterial luciferase.
Another luciferase system that has been used commercially for analytical purposes is the Aequorin system. The purified jellyfish photoprotein, aequorin, is used to detect and quantify intracellular Ca2+and its changes under various experimental conditions. The Aequorin photoprotein is relatively small [xcx9c20 kDa], nontoxic, and can be injected into cells in quantities adequate to detect calcium over a large concentration range [3xc3x9710xe2x88x927 to 10xe2x88x924 M].
Because of their analytical utility, luciferases and substrates have been studied and well-characterized and are commercially available [e.g., firefly luciferase is available from Sigma, St. Louis, Mo., and Boehringer Mannheim Biochemicals, Indianapolis, Ind.; recombinantly produced firefly luciferase and other reagents based on this gene or for use with this protein are available from Promega Corporation, Madison, Wis.; the aequorin photoprotein luciferase from jellyfish and luciferase from Renilla are commercially available from Sealite Sciences, Bogart, Ga.; coelenterazine, the naturally-occurring substrate for these luciferases, is available from Molecular Probes, Eugene, Oreg.]. These luciferases and related reagents are used as reagents for diagnostics, quality control, environmental testing and other such analyses.
Because of the utility of luciferases as reagents in analytical systems and the potential for use in high throughput screening systems, there is a need to identify and isolated a variety of luciferases that have improved or different spectral properties compared to those presently available. For all these reasons, it would be advantageous to have luciferases from a variety of species, such as Gaussia and various Renilla species available.
Fluorescent Proteins
Reporter genes, when co-transfected into recipient cells with a gene of interest, provide a means to detect transfection and other events. Among reporter genes are those that encode fluorescent proteins. The bioluminescence generating systems described herein are among those used as reporter genes. To increase the sensitivity bioluminescence generating systems have been combined with fluorescent compounds and proteins, such as naturally fluorescent phycobiliproteins. Also of interest are the fluorescent proteins that are present in a variety of marine invertebrates, such as the green and blue fluorescent proteins, particularly the green fluorescent protein (GFP) of Aequorea Victoria. 
The green fluorescent proteins (GFP) constitute a class of chromoproteins found only among certain bioluminescent coelenterates. These accessory proteins are fluorescent and function as the ultimate bioluminescence emitter in these organisms by accepting energy from enzyme-bound, excited-state oxyluciferin (e.g., see Ward et al. (1979) J. Biol. Chem. 254:781-788; Ward et al. (1978) Photochem. Photobiol. 27:389-396; Ward et al. (1982) Biochemistry 21:4535-4540).
The best characterized GFPs are those isolated from the jellyfish species Aequorea, particularly Aequorea Victoria (A. Victoria) and Aequorea forskxc3xa5lea (Ward et al. (1982) Biochemistry 21:4535-4540; Prendergast et al. (1978) Biochemistry 17:3448-3453). Purified A. victoria GFP is a monomeric protein of about 27 kDa that absorbs blue light with excitation wavelength maximum of 395 nm, with a minor peak at 470 nm, and emits green fluorescence with an emission wavelength of about 510 nm and a minor peak near 540 nm (Ward et al. (1979) Photochem. Photobiol. Rev 4:1-57),. This GFP has certain limitations. The excitation maximum of the wild Type GFP is not within the range of wavelengths of standard fluorescein detection optics.
The detection of green fluorescence does not require any 20 exogenous substrates or co-factors. Instead, the high level of fluorescence results from the intrinsic chromophore of the protein. The chromophore includes modified amino acid residues within the polypeptide chain. For example, the fluorescent chromophore of A. victoria GFP is encoded by the hexapeptide sequence, FSYGVQ, encompassing amino acid residues 64-69. The chromophore is formed by the intramolecular cyclization of the polypeptide backbone at residues Ser 65-, -Gly 67 and the oxidation of the xcex1-xcex2 bond of residue Tyr66(e.g., see Cody et al. (1993) Biochemistry 32:1212-1218; Shimomura (1978) FEBS Letters 104:220-222; Ward et al. (1989) Photochem. Photobiol. 49Ser; 25S). The emission spectrum of the isolated chromophore and the denatured protein at neutral pH do not match the spectrum of the native protein, suggesting that chromophore formation occurs post-translaionally (e.g., see Cody et al. (1993) Biochemistry 32:1212-1218).
In addition, the crystal structure of purified A. victoria GFP has been determined (e.g., see Ormxc3x6 (1996) Science 273:1392-1395). The predominant structural features of the protein are an 11-stranded xcex2 barrel that forms a nearly perfect cylinder wrapping around a single central xcex1-helix, which contains the modified p-hydroxybenzylideneimadaxolidinone chromophore. The chromophore is centrally located within the barrel structure and is completely shielded from exposure to bulk solvent.
DNA encoding an isotype of A. victoria GFP has been isolated and its nucleotide sequence has been determined (e.g., see Prasher (1992) Gene 111:229-233). The A. victoria cDNA contains a 714 nucleotide open reading frame that encodes a 238 amino acid polypeptide of a calculated Mr of 26,888 Da. Recombinantly expressed A. victoria GFPs retain their ability to fluoresce in vivo in a wide variety organisms, including bacteria (e.g., see Chalfie et al. (1994) Science 263:802-805; Miller et al. (1997) Gene 191:149-153), yeast and fungi (Fey et al. (1995) Gene 165:127-130; Straight et al. (1996) Curr. Biol. 6:1599-1608; Cormack et al. (1997) Microbiology 143:303-311), Drosophila (e.g., see Wang et al. (1994) Nature 369:400-403; Plautz (1996) Gene 173:83-87), plants (Heinlein et al. (1995); Casper et al. (1996) Gene 173:69-73), fish (Amsterdam et al. (1995) ), and mammals (Ikawa et al. (1995). Aequorea GFP vectors and isolated Aequorea GFP proteins have been used as markers for measuring gene expression, cell migration and localization, microtubule formation and assembly of functional ion channels (e.g., see Terry et al. (1995) Biochem. Biophys. Res. Commun. 217:21-27; Kain et al. (1995) Biotechnigues 19:650-655). The A. victoria GFP, however, is not ideal for use in analytical and diagnostic processes. Consequently GFP mutants have been selected with the hope of identifying mutants that have single excitation spectral peaks shifted to the red.
In fact a stated purpose in constructing such mutants has been to attempt to make the A. victoria GFP more like the GFP from Renilla, which has thus far not been cloned, but which has properties that make it far more ideal for use as an analytical tool. For many practical applications, the spectrum of Renilla GFP would be preferable to that of the Aequorea GFP, because wavelength discrimination between different fluorophores and detection of resonance energy transfer are easier if the component spectra are tall and narrow rather than low and broad [see, U.S. Pat. No. 5,625,048]. Furthermore, the longer wavelength excitation peak (475 nm) of Renilla GFP is almost ideal for fluorescein filter sets and is resistant to photobleaching, but has lower amplitude than the shorter wavelength peak at 395 nm, which is more susceptible to photobleaching [Chalfie et al. (1994) Science 263:802-805].
There exists a phylogenetically diverse and largely unexplored repertoire of bioluminescent proteins that are a reservoir for future development. Many of these, such as nucleic acid encoding Renilla GFPs have not, despite concentrated efforts to do so.
For these reasons, it would be desirable to have a variety of new luciferases and fluorescent proteins, particularly, Renilla GFP available rather than use muteins of A. victoria GFP. It has not, however, been possible to clone the gene encoding any Renilla GFPs. It would also be desirable to have a variety of GFPs and luciferases available in order to optimize systems for particular applications and to improve upon existing methods. Therefore, it is an object herein to provide isolated nucleic acids encoding heretofore unavailable luciferases and the protein encoded thereby. It is also an object herein to provide isolated nucleic acids encoding Renilla GFPs, GFPs from other species, and luciferases from a variety of species, and the proteins encoded thereby. It is also an object herein to provide bioluminescence generating systems that include the luciferases, luciferins, and also include GFPs.
Isolated nucleic acids that encode fluorescent proteins and nucleic acids that encode luciferases are provided. Nucleic acid molecules encoding GFPs from Renilla and from Ptilosarcus are provided. Nucleic acid molecules that encode the Renilla mulleri luciferase, a Gaussia species luciferase and a Pleuromamma species luciferase are provided. Nucleic acid probes derived therefrom are also provided. Functionally equivalent nucleic acids, such as those that hybridize under conditions of high stringency to the disclosed molecules, are also contemplated.
Host cells, including bacterial, yeast and mammalian host cells, and plasmids for expression of the nucleic acids encoding each luciferase and GFP and combinations of luciferases and GFPs are also provided in these hosts are also provided. The genes can be modified by substitution of codons optimized for expression in selected host cells or hosts, such as humans and other mammals, or can be mutagenized to alter the emission properties.
Luciferases
Recombinant host cells, including bacterial, yeast and mammalian cells, containing heterologous nucleic acid encoding a Renilla mulleri luciferase and the nucleic acid are provided. In preferred embodiments, the heterologous nucleic acid encodes the sequence of amino acids set forth in SEQ ID No. 18. In more preferred embodiments, the heterologous nucleic acid encodes the sequence of nucleotides set forth in SEQ ID No. 1 7. Also provided are functionally equivalent nucleic acids, such as nucleic acid molecules that hybridize under moderate or high stringency to the sequence of nucleotides set forth in SEQ ID No. 17, particularly when using the probes provided herein.
Isolated nucleic acids that encode luciferases from Gaussia are provided herein. In particular, nucleic acid fragments that encode Gaussia princeps luciferase, and nucleic acid probes derived therefrom are provided. In a particular embodiment, the luciferase is encoded by the sequence of nucleotides set forth in SEQ ID No. 19. Also provided are nucleic acid molecules that hybridize under moderate or high stringency to the sequence of nucleotides set forth in SEQ ID No. 19, particularly when using probes provided herein. Probes derived from this nucleic acid that can be used in methods provided herein to isolate luciferases from any Gaussia species are provided. In an exemplary embodiment, nucleic acid encoding Gaussia princeps luciferase is provided. This nucleic acid encodes the sequence of amino acids set forth in SEQ ID No. 20.
Nucleic acids that encode Pleuromamma luciferase are provided. In particular, a nucleic acid molecule that encodes a Pleuromamma luciferase and the encoded luciferase are set forth in SEQ ID Nos. 28 and 29, respectively. Nucleic acid encoding a Pleuromamma luciferase has also been isolated.
Expression vectors that contain DNA encoding a Renilla mulleri, Gaussia or Pleuromamma luciferase linked in operational association with a promoter element that allows for the constitutive or inducible expression of the luciferase are provided. In preferred embodiments, the vectors are capable of expressing the Renilla mulleri luciferase in a wide variety of host cells. Vectors for producing chimeric Renilla mulleri luciferase fusion proteins, preferably chimeric antibody-luciferase or acetylcholine esterase fusion proteins, containing a promoter element and a multiple cloning site located upstream or downstream of DNA encoding Renilla mulleri luciferase are also provided.
Recombinant cells containing heterologous nucleic acid encoding a Gaussia luciferase are also provided. Purified Gaussia luciferases and compositions containing a Gaussia luciferase alone or in combination with at other components of a bioluminescence-generating system, such as a Renilla green fluorescent protein, are provided. The Gaussia luciferase can be used, for example, to provide fluorescent illumination of novelty items or used in methods of detecting and visualizing neoplastic tissue and other tissues, detecting infectious agents using immunoassays, such homogenous immunoassays and in vitro fluorescent-based screening assays using multi-well assay devices, or provided in kits for carrying out any of the above-described methods. In particular, the Gaussia luciferase may be used in conjunction with suitable fluorescent proteins in assays provided herein.
Methods using the probes for the isolation and cloning of luciferase-encoding DNA in Gaussia, Pleuromamma and other species are also provided. In preferred embodiments, the nucleic acid probes are degenerate probes of at least 14 nucleotides, preferably 16 to 30 nucleotides, that are based on amino acids set forth in SEQ ID No. 19 and or the sequence of nucleotides set forth in SEQ ID No. 29.
Vectors containing DNA encoding a Gaussia luciferase or Pleuromamma luciferase are provided. In particular, expression vectors that contain DNA encoding the luciferase linked in operational association with a promoter element that allows for the constitutive or inducible expression of luciferase are provided. In preferred embodiments, the vectors are capable of expressing the luciferase in a wide variety of host cells. Vectors for producing chimeric luciferase fusion proteins (see, e.g., U.S. Pat. No. 5,464,745, which describes the use of protein binding domains; see SEQ ID Nos. 21 and 22, which set forth the sequences of a cellulose binding domain-luciferase fusion protein; and which are depicted in FIGS. 1 and 2) containing a promoter element and a multiple cloning site located upstream or downstream from DNA encoding Gaussia or Pleuromamma luciferase are also provided. In a particular embodiment, DNA encoding the luciferase is linked to DNA encoding the N-terminal portion of the cellulose binding domain (CBDclos; see, SEQ ID Nos. 21 and 22) in a PET vector (Novagen; see, U.S. Pat. Nos. 5,719,044 and 5,738,984, 5,670,623 and 5,496,934 and the Novagen catalog; complete sequences of each PET vector are provided with purchase of the vector).
Fusions of the nucleic acid, particularly DNA, encoding a Gaussia or Pleuromamma luciferase with DNA encoding a GFP or phycobiliprotein are also provided herein. Also provided are fusions of Renilla luciferase and a Renilla GFP. In these fusions the luciferase and GFP encoding DNA can be contiguous or separated by a spacer peptide. The fusions are used to produce fusion proteins, which by virtue of the interaction between the luciferase and GFP pair have a variety of unique analytical applications. The interaction is assessed by the emission spectrum of the luciferase-GFP protein pair in the presence of a luciferin and appropriate binding factors.
Recombinant host cells containing heterologous nucleic acid encoding a Gaussia or Pleuromamma luciferase are provided. In certain embodiments, the recombinant cells that contain the heterologous DNA encoding the luciferase are produced by transfection with DNA encoding a luciferase or by introduction of RNA transcripts of DNA encoding the protein. The DNA may be introduced as a linear DNA fragment or may be included in an expression vector for stable or transient expression of the encoding DNA.
The cells that express functional luciferase may be used alone or in conjunction with a bioluminescence-generating system, in cell-based assays and screening methods, such as those described herein. Presently preferred host cells for expressing the luciferase are bacteria, yeasts, fungi, plant cells, insect cells and animal cells.
Purified Gaussia, Pleuromamma and Renilla mulleri luciferases are provided. These luciferases are preferably obtained by expression of the nucleic acid provided herein in prokaryotic or eukaryotic cells that contain the nucleic acid that encodes the luciferase protein; and isolation of the expressed protein.
Compositions containing the luciferases are provided. The compositions can take any of a number of forms, depending on the intended method of use therefor. In certain embodiments, for example, the compositions contain a Gaussia luciferase, Gaussia luciferase peptide or Gaussia luciferase fusion protein, formulated for use in luminescent novelty items, immunoassays, donors in FET [fluorescent energy transfer] assays, FRET [fluorescent resonance energy transfer] assays, HTRF [homogeneous time-resolved fluorescence] assays or used in conjunction with multi-well assay devices containing integrated photodetectors, such as those described herein.
In more preferred embodiments, the bioluminescence-generating system includes, in addition to the luciferase a Renilla mulleri or Ptilosarcus GFP. These compositions can be used in a variety of methods and systems, such as included in conjunction with diagnostic systems for the in vivo detection of neoplastic tissues and other tissues, such as those methods described herein.
Combinations containing a first composition containing a luciferase and a second composition containing one or more additional components of a bioluminescence-generating system for use with articles of manufacture to produce novelty items are provided. These novelty items are designed for entertainment, recreation and amusement, and include, but are not limited to: toys, particularly squirt guns, toy cigarettes, toy xe2x80x9cHalloweenxe2x80x9d eggs, footbags and board/card games; finger paints and other paints, slimy play material; textiles, particularly clothing, such as shirts, hats and sports gear suits, threads and yarns; bubbles in bubble making toys and other toys that produce bubbles; balloons; figurines; personal items, such as cosmetics, bath powders, body lotions, gels, powders and creams, nail polishes, make-up, toothpastes and other dentifrices, soaps, body paints, and bubble bath; items such as inks, paper; foods, such as gelatins, icings and frostings; fish food containing luciferins and transgenic fish, particularly transgenic fish that express a luciferase; plant food containing a luciferin or luciferase, preferably a luciferin for use with transgenic plants that express luciferase; and beverages, such as beer, wine, champagne, soft drinks, and ice cubes and ice in other configurations; fountains, including liquid xe2x80x9cfireworksxe2x80x9d and other such jets or sprays or aerosols of compositions that are solutions, mixtures, suspensions, powders, pastes, particles or other suitable form.
Any article of manufacture that can be combined with a bioluminescence-generating system as provided herein and thereby provide entertainment, recreation and/or amusement, including use of the items for recreation or to attract attention, such as for advertising goods and/or services that are associated with a logo or trademark is contemplated herein. Such uses may be in addition to or in conjunction with or in place of the ordinary or normal use of such items. As a result of the combination, the items glow or produce, such as in the case of squirt guns and fountains, a glowing fluid or spray of liquid or particles. The novelty in the novelty item derives from its bioluminescence.
GFPS
Isolated nucleic acids that encode GFPs from Renilla are provided herein. Also provided are isolated and purified nucleic acids that encode a component of the bioluminescence generating system and a green fluorescent protein (GFP) of a member of the genus Renilla, and the proteins encoded thereby are provided. In particular, nucleic acid fragments that encode Renilla green fluorescent protein (GFPs) and the Renilla mulleri luciferase, and nucleic acid probes derived therefrom are provided.
Nucleic acid molecules encoding Renilla GFP are provided. In particular, nucleic acid molecules encoding a Renilla GFP that includes the coding portion of the sequence of nucleotides set forth in SEQ ID No. 15 or that hybridizes under moderate or high stringency to the sequence of nucleotides set forth in SEQ ID No. 15, particularly when using probes provided herein, are provided. Probes derived from this nucleic acid that can be used in methods provided herein to isolated GFPs from any Renilla species. In an exemplary embodiment, nucleic acid encoding Renilla mulleri GFP is provided. This nucleic acid encodes the sequence of amino acids set forth in SEQ ID No. 16.
Nucleic acid probes can be labeled, which if needed, for detection, containing at least about 14, preferably at least about 16, or, if desired, 20 or 30 or more, contiguous nucleotides of sequence of nucleotides encoding a Renilla GFP, particularly Renilla mulleri. In preferred embodiments, the nucleic acid probes for the Renilla GFP are selected from the sequence of nucleotides set forth in SEQ ID No. 15.
Methods using the probes for the isolation and cloning of GFP-encoding DNA in Renilla and other species are also provided. In preferred embodiments, the nucleic acid probes are degenerate probes based upon the conserved regions between the Renilla species of GFP as set forth in FIG. 3. Such degenerate probes contain at least 14 nucleotides, preferably 16 to 30 nucleotides, that are based on amino acids 51 to 68, 82 to 98 and 198 to 208 set forth in SEQ ID No. 16, amino acid sequence set forth in SEQ ID No. 24, amino acids 9-20 set forth in SEQ ID No. 25 and amino acids 39-53 set forth in SEQ ID No. 27. In other preferred embodiments, the nucleic acid probes encoding the above-described preferred amino acid regions are selected among the sequence of nucleotides encoding these regions as set forth in SEQ ID NO. 15. Alternatively, nucleic acids, particularly those set forth in SEQ ID No. 15 that encode the noted regions may be used as primers for PCR amplification of libraries of a selected Renilla species, whereby DNA comprising that encodes a Renilla GFP is isolated.
Nucleic acids that encode a Ptilosarcus GFP are set forth in SEQ ID Nos. 30 and 31; the encoded GFP is set forth in SEQ ID No. 32. Also provided are nucleic acid molecules that hybridize under moderate or high stringency to the sequence of nucleotides set forth in SEQ ID Nos. 28, 30 and 31.
Vectors containing DNA encoding a Renilla or Ptilosarcus GFP are provided. In particular, expression vectors that contain DNA encoding a Renilla or Ptilosarcus GFP linked in operational association with a promoter element that allows for the constitutive or inducible expression of Renilla or Ptilosarcus GFP are provided. Native Renilla GFP has been expressed.
The vectors are capable of expressing the Renilla GFP in a wide variety of host cells. Vectors for producing chimeric Renilla GFP fusion proteins containing a promoter element and a multiple cloning site located upstream or downstream of DNA encoding Renilla GFP are also provided.
Recombinant cells containing heterologous nucleic acid encoding a Ptilosarcus GFP, Renilla GFP, Renilla mulleri luciferase, Gaussia luciferase, and Pleuromamma luciferase are also provided. Purified Renilla mulleri GFP, Renilla reniformis GFP peptides and compositions containing a Renilla GFPs and GFP peptides alone or in combination with at least one component of a bioluminescence-generating system, such as a Renilla mulleri luciferase, are provided. The Renilla GFP and GFP peptide compositions can be used, for example, to provide fluorescent illumination of novelty items or used in methods of detecting and visualizing neoplastic tissue and other tissues, detecting infectious agents using immunoassays, such homogenous immunoassays and in vitro fluorescent-based screening assays using multi-well assay devices, or provided in kits for carrying out any of the above-described methods. In particular, these proteins may be used in FP [fluorescence polarization] assays, FET [fluorescent energy transfer] assays, FRET [fluorescent resonance energy transfer] assays and HTRF [homogeneous time-resolved fluorescence] assays and also in the BRET assays and sensors provided herein.
Non-radioactive energy transfer reactions, such as FET or FRET, FP and HTRF assays, are homogeneous luminescence assays based on energy transfer are carried out between a donor luminescent label and an acceptor label [see, e.g., Cardullo et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:8790-8794; Peerce et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83:8092-8096; U.S. Pat. No. 4,777,128; U.S. Pat. No. 5,162,508; U.S. Pat. No. 4,927,923; U.S. Pat. No. 5,279,943; and International PCT Application No. WO 92/01225]. Non-radioactive energy transfer reactions using GFPs have been developed [see, International PCT application Nos. WO 98/02571 and WO 97/28261]. Non-radioactive energy transfer reactions using GFPs and luciferases, such as a luciferase and its cognate GFP (or multimers thereof), such as in a fusion protein, are contemplated herein.
Nucleic acids that exhibit substantial sequence identity with the nucleic acids provided herein are also contemplated. These are nucleic acids that can be produced by substituting codons that encode conservative amino acids and also nucleic acids that exhibit at least about 80%, preferably 90 or 95% sequence identity. Sequence identity refers to identity as determined using standard programs with default gap penalties and other defaults as provided by the manufacturer thereof.
The nucleic acids provide an opportunity to produce luciferases and GFPs, which have advantageous application in all areas in which luciferase/luciferins and GFPs have application. The nucleic acids can be used to obtain and produce GFPs and GFPs from other, particularly Renilla species using the probes described herein that correspond to conserved regions (see, e.g., FIG. 3). These GFPs have advantageous application in all areas in which GFPs and/or luciferase/luciferins have application. For example, The GFP""s provide a means to amplify the output signal of bioluminescence generating systems. Renilla GFP has a single excitation absorbance peak in blue light (and around 498 nm) and a predominantly single emission peak around 510 nm (with a small shoulder near 540). This spectra provides a means for it to absorb blue light and efficiently convert it to green light. This results in an amplification of the output. When used in conjunction with a bioluminescence generating system that yields blue light, such as Aequorea or Renilla or Vargula (Cypridina), the output signal for any application, including diagnostic applications, is amplified. In addition, this green light can serve as an energy donor in fluorescence-based assays, such as fluorescence polarization assays, FET [fluorescent energy transfer] assays, FRET [fluorescent resonance energy transfer] assays and HTRF [homogeneous time-resolved fluorescence] assays. Particular assays, herein referred to as BRET [bioluminescence resonance energy transfer assays in which energy is transferred from a bioluminescence reaction of a luciferase to a fluorescent protein], are provided.
Non-radioactive energy transfer reactions, such as FET or FRET, FP and HTRF assays, are homogeneous luminescence assays based on energy transfer that are carried out between a donor luminescent label and an acceptor label [see, e.g., Cardullo et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:8790-8794; Peerce et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83:8092-8096; U.S. Pat. No. 4,777,128; U.S. Pat. No. 5,162,508; U.S. Pat. No. 4,927,923; U.S. Pat. No. 5,279,943; and International PCT Application No. WO 92/01225]. Non-radioactive energy transfer reactions using GFPs have been developed [see, International PCT application Nos. WO 98/02571 and WO 97/28261].
Mutagenesis of the GFPs is contemplated herein, particularly mutagenesis that results in modified GFPs that have red-shifted excitation and emission spectra. The resulting systems have higher output compared to the unmutagenized forms. These GFPs may be selected by random mutagenesis and selection for GFPs with altered spectra or by selected mutagenesis of the chromophore region of the GFP.
Recombinant host cells containing heterologous nucleic acid encoding a Renilla or Ptilosarcus GFP are also provided. In certain embodiments, the recombinant cells that contain the heterologous DNA encoding the Renilla or Ptilosarcus GFP are produced by transfection with DNA encoding a Renilla or Ptilosarcus GFP or by introduction of RNA transcripts of DNA encoding a Renilla or Ptilosarcus protein. The DNA may be introduced as a linear DNA fragment or may be included in an expression vector for stable or transient expression of the encoding DNA.
In certain embodiments, the cells contain DNA or RNA encoding a Renilla mulleri GFP or a Ptilosarcus GFP (particularly from a species other than P. gurneyi) also express the recombinant Renilla mulleri GFP or Ptilosarcus polypeptide. It is preferred that the cells are selected to express functional GFPs that retain the ability to fluorescence and that are not toxic to the host cell. In some embodiments, cells may also include heterologous nucleic acid encoding a component of a bioluminescence-generating system, preferably a photoprotein or luciferase. In preferred embodiments, the nucleic acid encoding the bioluminescence-generating system component is isolated from the species Aequorea, Vargula, Pleuromamma, Ptilosarcus or Renilla. In more preferred embodiments, the bioluminescence-generating system component is a Renilla mulleri luciferase including the amino acid sequence set forth in SEQ ID No. 18 or the Pleuromamma luciferase set forth in SEQ ID No. 28, or the Gaussia luciferase set forth in SEQ ID No. 19.
The GFPs provided herein may be used in combination with any suitable bioluminescence generating system, but is preferably used in combination with a Renilla or Aequorea, Pleuromamma or Gaussia luciferase.
Purified Renilla GFPs, particularly Renilla mulleri GFP, and purified Renilla reniformis GFP peptides are provided. Presently preferred Renilla GFP for use in the compositions herein is Renilla mulleri GFP including the sequence of amino acids set forth in SEQ ID No. 16. Presently preferred Renilla reniformis GFP peptides are those containing the GFP peptides selected from the amino acid sequences set forth in SEQ ID Nos 19-23.
The Renilla GFP, GFP peptides and luciferase can be isolated from natural sources or isolated from a prokaryotic or eukaryotic cell transfected with nucleic acid that encodes the Renilla GFP and/or luciferase protein.
Fusions of the nucleic acid, particularly DNA, encoding Renilla or Ptilosarcus GFP with DNA encoding a luciferase are also provided herein.
The cells that express functional luciferase and/or GFP, which may be used alone or in conjunction with a bioluminescence-generating system, in cell-based assays and screening methods, such as those described herein.
Presently preferred host cells for expressing GFP and luciferase are bacteria, yeasts, fungi, plant cells, insect cells and animal cells.
The luciferases and GFPs or cells that express them also may be used in methods of screening for bacterial contamination and methods of screening for metal contaminants. To screen for bacterial contamination, bacterial cells that express the luciferase and/or GFP are put in autoclaves or in other areas in which testing is contemplated. After treatment or use of the area, the area is tested for the presence of glowing bacteria. Presence of such bacteria is indicative of a failure to eradicate other bacteria. Screening for heavy metals and other environmental contaminants can also be performed with cells that contain the nucleic acids provided herein, if expression is linked to a system that is dependent upon the particular heavy metal or contaminant.
The systems and cells provided herein can be used for high throughout screening protocols, intracellular assays, medical diagnostic assays, environmental testing, such as tracing bacteria in water supplies, in conjunction with enzymes for detecting heavy metals, in spores for testing autoclaves in hospital, foods and industrial autoclaves. Non-pathogenic bacteria containing the systems can be included in feed to animals to detect bacterial contamination in animal products and in meats.
Compositions containing a Renilla or Ptilosarcus GFP are provided. The compositions can take any of a number of forms, depending on the intended method of use therefor. In certain embodiments, for example, the compositions contain a Renilla GFP or GFP peptide, preferably Renilla mulleri GFP or Renilla reniformis GFP peptide, formulated for use in luminescent novelty items, immunoassays, FET [fluorescent energy transfer] assays, FRET [fluorescent resonance energy transfer] assays, HTRF [homogeneous time-resolved fluorescence] assays or used in conjunction with multi-well assay devices containing integrated photodetectors, such as those described herein. In other instances, the GFPs are used in beverages, foods or cosmetics.
Compositions that contain a Renilla mulleri GFP or GFP peptide and at least one component of a bioluminescence-generating system, preferably a luciferase, luciferin or a luciferase and a luciferin, are provided. In preferred embodiments, the luciferase/luciferin bioluminescence-generating system is selected from those isolated from: an insect system, a coelenterate system, a ctenophore system, a bacterial system, a mollusk system, a crustacea system, a fish system, an annelid system, and an earthworm system. Bioluminescence-generating systems include those isolated from Renilla, Aequorea, and Vargula, Gaussia and Pleuromamma.
Combinations containing a first composition containing a Renilla mulleri GFP or Ptilosarcus GFP or mixtures thereof and a second composition containing a bioluminescence-generating system for use with inanimate articles of manufacture to produce novelty items are provided. These novelty items, which are articles of manufacture, are designed for entertainment, recreation and amusement, and include, but are not limited to: toys, particularly squirt guns, toy cigarettes, toy xe2x80x9cHalloweenxe2x80x9d eggs, footbags and board/card games; finger paints and other paints, slimy play material; textiles, particularly clothing, such as shirts, hats and sports gear suits, threads and yarns; bubbles in bubble making toys and other toys that produce bubbles; balloons; figurines; personal items, such as bath powders, body lotions, gels, powders and creams, nail polishes, cosmetics including make-up, toothpastes and other dentifrices, soaps, cosmetics, body paints, and bubble bath, bubbles made from non-detergent sources, particularly proteins such as albumin and other non-toxic proteins; in fishing lures, particularly cross-linked polyacrylamide containing a fluorescent protein and/or components of a bioluminescence generating system, which glow upon contact with water; items such as inks, paper; foods, such as gelatins, icings and frostings; fish food containing luciferins and transgenic fish, particularly transgenic fish that express a luciferase; plant food containing a luciferin or luciferase, preferably a luciferin for use with transgenic plants that express luciferase; and beverages, such as beer, wine, champagne, soft drinks, and ice cubes and ice in other configurations; fountains, including liquid xe2x80x9cfireworksxe2x80x9d and other such jets or sprays or aerosols of compositions that are solutions, mixtures, suspensions, powders, pastes, particles or other suitable form.
Any article of manufacture that can be combined with a bioluminescence-generating system as provided herein and thereby provide entertainment, recreation and/or amusement, including use of the items for recreation or to attract attention, such as for advertising goods and/or services that are associated with a logo or trademark is contemplated herein. Such uses may be in addition to or in conjunction with or in place of the ordinary or normal use of such items. As a result of the combination, the items glow or produce, such as in the case of squirt guns and fountains, a glowing fluid or spray of liquid or particles.
Methods for diagnosis and visualization of tissues in vivo or in situ using compositions containing a Renilla mulleri GFP and/or a Renilla mulleri luciferase or others of the luciferases and/or GFPs provided herein are provided. For example, the Renilla mulleri GFP protein can be used in conjunction with diagnostic systems that rely on bioluminescence for visualizing tissues in situ. The systems are particularly useful for visualizing and detecting neoplastic tissue and specialty tissue, such as during non-invasive and invasive procedures. The systems include compositions containing conjugates that include a tissue specific, particularly a tumor-specific, targeting agent linked to a targeted agent, a Renilla mulleri GFP, a luciferase or luciferin. The systems also include a second composition that contains the remaining components of a bioluminescence generating reaction and/or the Renilla mulleri GFP. In some embodiments, all components, except for activators, which are provided in situ or are present in the body or tissue, are included in a single composition.
Methods for diagnosis and visualization of tissues in vivo or in situ using compositions containing a Gaussia luciferase are provided. For example, the Gaussia luciferase or Gaussia luciferase peptide can be used in conjunction with diagnostic systems that rely on bioluminescence for visualizing tissues in situ. The systems are particularly useful for visualizing and detecting neoplastic tissue and specialty tissue, such as during non-invasive and invasive procedures. The systems include compositions containing conjugates that include a tissue specific, particularly a tumor-specific, targeting agent linked to a targeted agent, a Gaussia luciferase, a GFP or luciferin. The systems also include a second composition that contains the remaining components of a bioluminescence generating reaction and/or the Gaussia luciferase. In some embodiments, all components, except for activators, which are provided in situ or are present in the body or tissue, are included in a single composition.
In particular, the diagnostic systems include two compositions. A first composition that contains conjugates that, in preferred embodiments, include antibodies directed against tumor antigens conjugated to a component of the bioluminescence generating reaction, a luciferase or luciferin, preferably a luciferase are provided. In certain embodiments, conjugates containing tumor-specific targeting agents are linked to luciferases or luciferins. In other embodiments, tumor-specific targeting agents are linked to microcarriers that are coupled with, preferably more than one of the bioluminescence generating components, preferably more than one luciferase molecule.
The second composition contains the remaining components of a bioluminescence generating system, typically the luciferin or luciferase substrate. In some embodiments, these components, particularly the luciferin are linked to a protein, such as a serum albumin, or other protein carrier. The carrier and time release formulations, permit systemically administered components to travel to the targeted tissue without interaction with blood cell components, such as hemoglobin that deactivates the luciferin or luciferase.
Methods for diagnosing diseases, particularly infectious diseases, using chip methodology (see, e.g., copending U.S. application Ser. No. 08/990,103) a luciferase/luciferin bioluminescence-generating system and a Renilla mulleri or Ptilosarcus GFP are provided. In particular, the chip includes an integrated photodetector that detects the photons emitted by the bioluminescence-generating system, particularly using luciferase encoded by the nucleic acids provided herein and/or Renilla mulleri or Ptilosarcus GFP.
In one embodiment, the chip is made using an integrated circuit with an array, such as an X-Y array, of photodetectors. The surface of circuit is treated to render it inert to conditions of the diagnostic assays for which the chip is intended, and is adapted, such as by derivatization for linking molecules, such as antibodies. A selected antibody or panel of antibodies, such as an antibody specific for a bacterial antigen, is affixed to the surface of the chip above each photodetector. After contacting the chip with a test sample, the chip is contacted with a second antibody linked to a Renilla or Pleuromamm GFP, a chimeric antibody-Renilla GFP fusion protein or an antibody linked to a component of a bioluminescence generating system, such as a luciferase or luciferin, that are specific for the antigen. The remaining components of the bioluminescence generating reaction are added, and, if any of the antibodies linked to a component of a bioluminescence generating system are present on the chip, light will be generated and detected by the adjacent photodetector. The photodetector is operatively linked to a computer, which is programmed with information identifying the linked antibodies, records the event, and thereby identifies antigens present in the test sample.
Methods for generating chimeric GFP fusion proteins are provided. The methods include linking DNA encoding a gene of interest, or portion thereof, to DNA encoding a GFP coding region in the same translational reading frame. The encoded-protein of interest may be linked in-frame to the amino- or carboxyl-terminus of the GFP. The DNA encoding the chimeric protein is then linked in operable association with a promoter element of a suitable expression vector. Alternatively, the promoter element can be obtained directly from the targeted gene of interest and the promoter-containing fragment linked upstream of the GFP coding sequence to produce chimeric GFP proteins.
Methods for identifying compounds using recombinant cells that express heterologous DNA encoding a GFP under the control of a promoter element of a gene of interest are provided. The recombinant cells can be used to identify novel compounds or ligands that modulate the level of transcription from the promoter of interest by measuring GFP-mediated fluorescence. Recombinant cells expressing the chimeric Renilla or Ptilosarcus GFPs may also be used for monitoring gene expression or protein trafficking, or determining the cellular localization of the target protein by identifying localized regions of GFP-mediated fluorescence within the recombinant cell.
Other assays using the GFPs and/or luciferases are contemplated herein. Any assay or diagnostic method known used by those of skill in the art that employ Aequora GFPs and/or other luciferases are contemplated herein.
Kits containing the GFPs for use in the methods, including those described herein, are provided. In one embodiment, the kits containing an article of manufacture and appropriate reagents for generating bioluminescence are provided. The kits containing such soap compositions, with preferably a moderate pH [between 5 and 8] and bioluminescence generating reagents, including luciferase and luciferin and the GFP are provided herein. These kits, for example, can be used with a bubble-blowing or producing toy. These kits can also include a reloading or charging cartridge or can be used in connection with a food.
In another embodiment, the kits are used for detecting and visualizing neoplastic tissue and other tissues and include a first composition that contains the GFP and at least one component of a bioluminescence generating system, and a second that contains the activating composition, which contains the remaining components of the bioluminescence generating system and any necessary activating agents.
Thus, these kits will typically include two compositions, a first composition containing the GFP formulated for systemic administration (or in some embodiments local or topical application), and a second composition containing the components or remaining components of a bioluminescence generating system, formulated for systemic, topical or local administration depending upon the application. Instructions for administration will be included.
In other embodiments, the kits are used for detecting and identifying diseases, particularly infectious diseases, using multi-well assay devices and include a multi-well assay device containing a plurality of wells, each having an integrated photodetector, to which an antibody or panel of antibodies specific for one or more infectious agents are attached, and composition containing a secondary antibody, such as an antibody specific for the infectious agent that is linked to a Renilla mulleri or Ptilosarcus GFP protein, a chimeric antibody-Renilla mulleri (or Ptilosarcus) GFP fusion protein or F(Ab)2 antibody fragment-Renilla mulleri GFP fusion protein. A second composition containing a bioluminescence generating system that emits a wavelength of light within the excitation range of the Renilla mulleri GFP, such as species of Renilla or Aequorea, for exciting the Renilla mulleri, which produces green light that is detected by the photodetector of the device to indicate the presence of the agent.
As noted above, fusions of nucleic acid encoding the luciferases and or GFPs provided herein with other luciferases and GFPs are provided. Of particular interest are fusions that encode pairs of luciferases and GFPs, such as a Renilla luciferase and a Renilla GFP (or a homodimer or other multiple of a Renilla GFP). The luciferase and GFP bind and in the presence of a luciferin will produced fluorescence that is red shifted compared to the luciferase in the absence of the GFP. This fusion or fusions in which the GFP and luciferase are linked via a target, such as a peptide, can be used as a tool to assess anything that interacts with the linker.
Muteins of the GFPs and luciferases are provided. Of particular interest are muteins, such as temperature sensitive muteins, of the GFP and luciferases that alter their interaction, such as mutations in the Renilla luciferase and Renilla GFP that alters their interaction at a critical temperature.
Antibodies, polyclonal and monoclonal antibodies that specifically bind to any of the proteins encoded by the nucleic acids provided herein are also provided. These antibodies, monoclonal or polyclonal, can be prepared employing standard techniques, known to those of skill in the art. In particular, immunoglobulins or antibodies obtained from the serum of an animal immunized with a substantially pure preparation of a luciferase or GFP provided herein or an or epitope-containing fragment thereof are provided. Monoclonal antibodies are also provided. The immunoglobulins that are produced have, among other properties, the ability to specifically and preferentially bind to and/or cause the immunoprecipitation of a GFP or luciferase, particularly a Renilla or Ptsilocarpus GFP or a Pleuromamma, Gaussia or Renilla mulleri luciferase, that may be present in a biological sample or a solution derived from such a biological sample.